Epidemiology of Paenibacillus larvae, causative agent of American foulbrood

135 Pages·2016·3.09 MB·English

by  Barbara Morrissey

Epidemiology of Paenibacillus larvae, causative agent
of American foulbrood

Barbara Joy Morrissey
PhD
University of York
Biology
September 2015
Abstract
Paenibacillus larvae is the causative agent of American foulbrood (AFB), a damaging
disease of honey bees (Apis mellifera) with a global distribution. AFB infects the honey
bee larvae through spore-contaminated larval food and once infected the larvae will die
within twelve days. AFB infection leads to the eventual death of the honey bee colony. In
many countries the best control method is thought to be the destruction of the colony, to
destroy the hardy, infectious spores. Therefore infection nearly always leads to the death of
the colony. In the UK cases of the disease have decreased in recent years due to statutory
control methods, however sporadic outbreaks occur each year. Many advances have been
made in our knowledge of the mechanisms of infection by P. larvae in recent years
however, a high resolution, standardised method of strain typing is required to track
disease spread and understand outbreak sources. This thesis describes the development of
the first multilocus sequence typing (MLST) scheme for P. larvae. MLST is the gold
standard for pathogenic bacteria typing. It is based on the sequencing of sections of 6-10
housekeeping genes. MLST is standardised, as primer and allele sequences can be made
available for other researchers. The new MLST scheme was used to describe previously
undetectable patterns of distribution at a global level as well as at a national level. Humans
and bees are implicated in the movement of the disease over different spatial scales. Using
the MLST scheme a diverse group of isolates were selected for whole genome sequencing.
For the first time multiple genomes were compared within and between genotypes. The
ability of the MLST scheme to describe relationships amongst sequence types (STs) was
tested by comparing phylogenies based on core genes and MLST sequences. I describe the
discovery of seven plasmids in four STs previously unknown to harbour plasmids.

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Table of Contents
Abstract ................................................................................................................. 2
Table of Contents ................................................................................................. 3
List of Tables ........................................................................................................ 7
List of Figures ....................................................................................................... 8
Acknowledgements ............................................................................................. 10
Author’s declaration .......................................................................................... 11
1 Introduction .................................................................................................. 12
1.1.1 Pathogenesis of AFB ................................................................................. 12
1.1.2 Spread of AFB ........................................................................................... 13
1.1.3 Control of AFB .......................................................................................... 13
1.1.4 AFB control in the UK .............................................................................. 14
1.1.5 Classification of Paenibacillus larvae ....................................................... 14
1.1.6 ERIC typing of P. larvae ........................................................................... 15
1.1.7 ERIC types I and II .................................................................................... 15
1.1.8 Strain typing methods for P. larvae and biogeography ............................. 16
1.1.9 MultiLocus Sequence Typing (MLST) ..................................................... 17
1.1.10 M. plutonius MLST scheme .................................................................... 18
1.1.11 Bacterial genome sequencing .................................................................. 18
1.1.12 P. larvae genomics .................................................................................. 19
1.1.13 P. larvae comparative genomics ............................................................. 19
1.1.14 Plasmids in P. larvae ............................................................................... 20
1.2 This thesis ........................................................................................................ 21
2 Biogeography of Paenibacillus larvae, the causative agent of American foulbrood,
using a new MLST scheme ................................................................................ 23
2.1 Abstract ........................................................................................................... 23
2.2 Introduction .................................................................................................... 23
2.2.1 AFB control ............................................................................................... 24
2.2.2 P. larvae genotyping .................................................................................. 24
2.2.3 MultiLocus Sequence Typing .................................................................... 25
2.3 Methods ........................................................................................................... 26
2.3.1 MLST development. .................................................................................. 26
2.3.2 Primer design. ............................................................................................ 26
2.3.3 Global isolates. .......................................................................................... 27
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2.3.4 Sequencing................................................................................................. 27
2.3.5 MLST Analysis. ......................................................................................... 27
2.3.6 Population structure and biogeography. .................................................... 28
2.4 Results .............................................................................................................. 29
2.4.1 Loci discovery and primer design. ............................................................. 29
2.4.2 Index of association ................................................................................... 32
2.4.3 Sequence types .......................................................................................... 32
2.4.4 Population structure and biogeography. .................................................... 33
2.4.5 Correspondence Analysis (CA). ................................................................ 37
2.4.6 Rarefaction. ................................................................................................ 37
2.5 Discussion ........................................................................................................ 40
2.5.1 P. larvae biogeography .............................................................................. 40
3 Patterns of distribution of the honey bee pathogen Paenibacillus larvae in the UK
45
3.1 Abstract ........................................................................................................... 45
3.2 Introduction .................................................................................................... 45
3.2.1 UK AFB control ........................................................................................ 46
3.2.2 AFB spread ................................................................................................ 46
3.2.3 MLST......................................................................................................... 47
3.3 Methods ........................................................................................................... 47
3.3.1 DNA Extraction. ........................................................................................ 48
3.3.2 Analyses..................................................................................................... 55
3.4 Results .............................................................................................................. 59
Sequence types. ...................................................................................................... 59
3.4.1 Clusters and risk points. ............................................................................. 59
3.4.2 Clusters and background............................................................................ 59
3.4.3 Comparison to global STs ......................................................................... 62
3.4.4 Honey samples. .......................................................................................... 62
3.5 Discussion ........................................................................................................ 62
4 Comparison of Paenibacillus larvae population structure determined using
multilocus sequence- typing and whole genome sequencing data ................. 68
4.1 Abstract ........................................................................................................... 68
4.2 Introduction .................................................................................................... 68
4.2.1 The bacterial genome ................................................................................. 69
4.2.2 Pan-genome ............................................................................................... 69
4.2.3 Paenibacillus larvae .................................................................................. 70
4.2.4 Genome Sequencing in P. larvae .............................................................. 70
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4.2.5 Genomic comparison of P. larvae genotypes ............................................ 70
4.3 Methods ........................................................................................................... 71
4.3.1 Bacterial culturing. .................................................................................... 71
4.3.2 Whole genome sequencing ........................................................................ 72
4.3.3 Genome assembly ...................................................................................... 72
4.3.4 Core genes and accessory genes. ............................................................... 74
4.4 Results .............................................................................................................. 75
4.4.1 MLST typing mistakes. ............................................................................. 75
4.4.2 Genome features ........................................................................................ 75
4.4.3 Phylogenies. ............................................................................................... 83
4.5 Discussion ........................................................................................................ 83
4.5.1 Phylogeny .................................................................................................. 83
4.5.2 Accessory genes ........................................................................................ 84
4.5.3 Phages ........................................................................................................ 85
4.5.4 Plasmids ..................................................................................................... 85
5 Description and phylogeny of Paenibacillus larvae plasmids discovered in rare
strains .................................................................................................................. 87
5.1 Abstract ........................................................................................................... 87
5.2 Introduction .................................................................................................... 87
5.2.1 Plasmid functions ...................................................................................... 88
5.2.2 Plasmids in P. larvae ................................................................................. 88
5.3 Methods ........................................................................................................... 89
5.4 Results .............................................................................................................. 92
5.4.1 Annotation ................................................................................................. 94
5.4.2 Comparison ................................................................................................ 94
5.4.3 Phylogeny .................................................................................................. 94
5.5 Discussion ........................................................................................................ 97
5.5.1 Movement and Phylogeny ......................................................................... 98
6 General Discussion ..................................................................................... 100
6.1 Background ................................................................................................... 100
6.2 Main findings of thesis ................................................................................. 101
6.3 Direction of future work .............................................................................. 103
6.3.1 ERIC I and II virulence............................................................................ 104
6.4 Conclusion ..................................................................................................... 105
7 Appendices .................................................................................................. 106
7.1 Appendix 1 MLST primer sequences tested and rejected ........................ 106
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7.2 Appendix 2 Origins of isolates typed in the development of the MLST scheme
107
8 References ................................................................................................... 118

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List of Tables
Table 2.1. MLST scheme primer sequences ........................................................ 30
Table 2.2 Feature summary of seven loci selected for P. larvae typing scheme . 31
Table 3.1 Data for UK P. larvae isolates typed in this chapter ........................... 49
Table 3.2 MLST allele numbers for UK P. larvae STs ....................................... 57
Table 3.3 Bray-Curtis test of dissimilarity results ............................................... 61
Table 3.4 P. larvae occurrence in UK imported honey samples ......................... 63
Table 4.1 P. larvae isolates selected for whole genome sequencing ................... 73
Table 4.2 General features of P. larvae genomes ................................................ 76
Table 4.3 Core and accessory genomes. .............................................................. 78
Table 5.1 General features of sequenced plasmids .............................................. 91
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List of Figures
Figure 2.1 eBURST diagram of P. larvae MLST scheme. Numbers represent ST of
isolates. STs with variations at more than three loci are not connected. Size of circles
represents the number of isolates of that type. Blue circles are typed as ERIC I, green
as ERIC II, purple as ERIC III, brown ERIC IV ......................................... 34
Figure 2.2 Neighbour joining tree inferring the evolutionary history of P. larvae STs. The
percentage of replicate trees in which the associated taxa clustered together in the
bootstrap test (1000 replicates) are shown next to the branches. ................. 35
Figure 2.3 Chao1 estimates of ERIC I and ERIC II ST richness as a function of sample
size. Dotted lines are 95% CIs and were calculated with the variance formula derived
by Chao (1987). The lower solid line represents ERIC II and the upper solid line
represents ERIC I ......................................................................................... 36
Figure 2.4 FST by distance. Figure 2.4A describes the F by distance of P larvae
ST
populations within the native range of the host (P=0.01). Figure 2.4B describes the
F by distance of P. larvae populations both within (dark grey dots) and outside the
ST
host's native range (light grey dots) (P= 0.996). .......................................... 38
Figure 2.5 Correspondence analysis ordination graph. The CA ordination graph illustrates
the association amongst countries of isolate origin and MLST STs. Filled circles
represent countries (dark grey represents native host range, light grey represents
introduced host range) Diamonds represent STs. ........................................ 39
Figure 3.2 Approximate location of AFB disease clusters with pie charts displaying ST
composition .................................................................................................. 60
Figure 4.1 Circle plots showing the genome sequences of ERIC I isolates compared to
reference sequence DSM 25719................................................................... 79
Figure 4.2 Circle plots showing the genome sequences of ERIC II isolates compared to
reference sequence DSM 25430................................................................... 80
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Figure 4.3 Neighbour net tree using concatenated MLST allele sequence data. The tree
shows the relationship between all 24 identified STs. ................................. 81
Figure 4.4 Neighbour net tree using core gene sequence data. The tree shows the
relationship between the 19 genome-sequenced isolates. The labels contain the ST,
Isolate name and number of non-core genes. ............................................... 82
Figure 5.1 Circle diagram of plasmids. ERIC I plasmids above, ERIC II plasmids below.
Regions with coverage more than one standard deviation from the mean coverage are
coloured blue regions, regions with coverage less than one standard deviation from
the mean are coloured red. ........................................................................... 93
Figure 5.2 Maximum Likelihood tree of plasmid sequences. The percentage of replicate
trees in which the associated taxa clustered together in the bootstrap test (1000
replicates) are shown next to the branches. .................................................. 95
Figure 5.3 Maximum Likelihood tree of isolates containing plasmids using core gene
sequences. The percentage of replicate trees in which the associated taxa clustered
together in the bootstrap test (1000 replicates) are shown next to the branches.
...................................................................................................................... 96

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Acknowledgements
My PhD was a BBSRC CASE studentship with Bee disease insurance. The work in
Chapters three and four was funded jointly by a grant from BBSRC, Defra, NERC, the
Scottish Government and the Wellcome Trust, under the Insect Pollinator Initiative
(BB/I000801/1); and a Defra/Fera seedcorn project (PI Budge). Firstly I’d like to thank my
supervisors Giles Budge and Thorunn Helgason for their help and support during my PhD.
I am grateful for their knowledge and enthusiasm and the advice they gave me during my
thesis. I would also like to thank Bernard Diaper for support, interest and commitment to
my work. Thanks to Ummey Hany for training me in next generation sequencing, which
formed an important part of my PhD. I would like to thank everyone at the National Bee
Unit, but especially Jan Bew and Victoria Tomkies for their help in the lab and Ed Haynes
and Ben Jones for discussing my work. Peter Ashton, Toby Hodges and Richard Randal-
Boggis were a great help whenever I had difficulties with bioinformatics and computer
programing.
I would like to thank my friends and family for their help and support throughout the
difficult process of writing up my thesis. Ruth, Kath, Clair, Cat and Farida – thanks for the
chance to escape and for all the gin. I would also like to thank Ian Cleasby for lots of help
during my PhD including helping with analysis, reading chapters and providing comments
on everything. I am also grateful for the help that Rufus provided, though he never said
much he always cheered me up. Finally, my parents were a great help throughout my PhD
and supported me throughout in numerous different ways. I hope this PhD makes them
proud.

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